Sibyl is a package designed to test different rarefaction thresholds when normalizing microbial abundance data from 16S amplicon sequencing. It has been created to answer a simple question:“How low can you go”?

Installation

The easiest way to install Sibyl is from our R-Universe, which provides prebuilt binaries for Windows and macOS, and source packages for Linux. Bioconductor repositories are added automatically for dependencies like phyloseq.

# Sibyl requires R >= 4.2
if (getRversion() < "4.2.0")
  stop("Sibyl requires R >= 4.2.0. Please update R and try again.", call. = FALSE)

# Add Bioconductor if missing
if (!requireNamespace("BiocManager", quietly = TRUE))
  install.packages("BiocManager", repos = "https://cloud.r-project.org")

# Prefer R-Universe + BioC
options(repos = c(
  headonpillow = "https://headonpillow.r-universe.dev",
  BiocManager::repositories(),
  CRAN = "https://cloud.r-project.org"
))

install.packages("Sibyl")
library(Sibyl)

Linux users: if you see errors about missing system libraries

Some dependencies may build from source on Linux. If installation fails, install the following system packages once and rerun the R code above.

Ubuntu/Debian

sudo apt update && sudo apt install -y \
  build-essential r-base-dev \
  libcurl4-openssl-dev libssl-dev libxml2-dev zlib1g-dev libgit2-dev \
  libjpeg-dev libpng-dev libx11-dev libglu1-mesa-dev freeglut3-dev libgl1-mesa-dev \
  liblapack-dev gfortran pkg-config

Fedora

sudo dnf install -y \
  R-devel @development-tools \
  libcurl-devel openssl-devel libxml2-devel zlib-devel libgit2-devel \
  libjpeg-turbo-devel libpng-devel libX11-devel mesa-libGLU-devel mesa-libGL-devel \
  lapack-devel blas-devel gcc-gfortran pkgconf-pkg-config

Why using Sibyl

The Sibyl package was created with the aim of testing the lower limit of rarefaction thresholds.

While microbial abundance data is slowly moving away from methods like rarefaction to account for differing library size, rarefaction has been extensively used and still in 2025 is present in much of the available literature, and still discussed.

When performing Principal Component Analysis (PCA) and other types of ordination, it is necessary making sure to choose a rarefaction threshold which does not impact the structure of the data, or our conclusions, during exploratory analysis. Usually, when chosing a rarefaction threshold researchers need to operate a compromise between describing samples accurately (completeness), and including more samples, which might sometimes not meet the selected threshold.

Sibyl aims to solve that, allowing users to explore sample completeness, and the effect of rarefaction thresholds on ordinations, all with a single tool.